There is convincing evidence that tobacco use, both as cigarettes and smokeless tobacco preparations, is highly dependence producing and that chronic use can result in tolerance. Although many behavioral factors are thought to be involved in the initiation and maintenance of tobacco use, there is also convincing evidence that nicotine is necessary for the dependence process to occur. Despite this evidence, little is known about the neurochemical effects of nicotine in the brain or about the neurochemical consequences of chronic nicotine exposure. Previous investigators have shown that chronic nicotine treatment has effects on the number of nicotinic cholinergic binding sites in the brain, but these results are somewhat contradictory. The aims of the studies outlined in this proposal are to use a comprehensive approach to describe the neurochemical effects of nicotine in the brain and to determine whether these effects are involved in nicotine tolerance. In specific, three aspects of the functioning of the CNS nicotinic receptor will be explored: (1) Nicotinic cholinergic receptor regulation of sodium flux across neuronal membranes will be studied by measuring 22Na influx into synaptosomes. (2) The influx of sodium through receptor operated channels is hypothesized to depolarize the membrane and open voltage-regulated calcium channels. Nicotine-induced changes in synaptosomal calcium flux will be studied using 45Ca. (3) Monoamines are known to be released by nicotinic agonists. The mechanism of this release will be studied, with particular attention to the involvement of sodium and calcium influxes as preceding events. In addition, experiments will be carried out to determine whether the effects of nicotine on sodium or calcium influx or neurotransmitter release are altered in animals chronically treated with nicotine. The hypothesis to be tested is that tolerance to nicotine is due to a functional "down regulation" of the nicotinic cholinergic receptor system. As these experiments are performed, the pattern of relationships among brain region, neurochemical changes and physiological and behavioral measures of tolerance will be studied in rats exposed to various doses of nicotine, carbachol and a cholinesterase inhibitor. It is hoped that a consistent pattern of changes will emerge that will increase our insight into the neurochemical actions of nicotine and will demonstrate whether long term nicotine use changes the functioning of the CNS nicotinic cholinergic receptor system.